This research investigates the relationship between the microstructure and mechanical properties of electron beam welding joints, with a particular emphasis on the influence of varying electron beam currents on AISI 410L stainless steel plates and NS163 Co-based superalloy wires, which is a rare and novel form of welding. An increase in beam current led to a gradual transformation of the weld microstructure from columnar to equiaxed grains, creating finer and more homogeneous grains. Macrosegregation was also observed, a consequence of the liquidus temperature difference between the weld metal (WM) and base metal (BM). The segregation structure exhibited irregularly shaped beaches near the fusion line and islands of unmixed and partially mixed NS163 within the WM. This phenomenon was more pronounced in samples with a lower beam current. Given the limitations in BM shape and WM size, nanoindentation was employed to characterize the strength of welded joints. In conjunction with microhardness measurements, it was confirmed that the weld is strengthened under different electron beam currents, with higher current resulting in a more significant strengthening effect. This study also underscores the value of nanoindentation in evaluating the weld strength of brush seals without destruction.